Nucleic acid encoding a novel ribonuclease having an amino acid sequence made up of the amino acid sequence of a known ribonuclease and an n-terminal leader sequence that is at least one residue long

ABSTRACT

A nucleic acid encodes a novel RNase. The RNase has an amino acid sequence in which an amino acid sequence disclosed in U.S. Pat. No. 6,239,257 B1 is preceded by a different N-terminal residue or leader sequence.

BACKGROUND OF THE INVENTION

The invention relates to pharmaceuticals, and more particularly relatesto pharmaceuticals for treating tumors in humans. In its most immediatesense, the invention relates to bioactive ribonucleases (“RNases”).

Some RNases are known to be active against certain human tumor cells.For example, commonly-owned U.S. Pat. No. 5,559,212 discloses and claimsranpirnase, an RNase pharmaceutical that is presently known by theregistered trademark ONCONASE and that is presently the subject of PhaseIII clinical trials. And, commonly-owned U.S. Pat. No. 6,239,257 B1discloses four RNase proteins that belong to the pancreatic RNase Asuperfamily, each possessing activity against two human carcinoma celllines.

Attention is now being directed to “targeting” pharmaceuticals todeliver them to particular cell receptors of interest. This isaccomplished by selecting a targeting moiety that is preferentiallyattracted to the desired cell receptor and attaching (as by conjugationor fusion) the targeting moiety to the pharmaceutical.

Commonly-owned U.S. Pat. No. 6,175,003 B1 discusses the concept oftargeting therapeutically active RNases by “cysteinizing” them. In thecase of ranpirnase, this can be accomplished by conjugating thetargeting moiety to the cysteine residue at position 72. While thisapproach is promising and is still under investigation, some peoplebelieve that it may be difficult to obtain regulatory approval for aconjugate and that a fusion protein would have an easier path toregulatory approval.

The N-terminal residue of ranpirnase is pyroglutamic acid. This “blocks”the N-terminal, i.e. makes it impossible to attach other amino acidresidues to the left of the N-terminal. For this reason, it is notpossible to create a fusion protein by attaching a targeting moiety tothe N-terminal of ranpirnase. And, while it is possible to remove thepyroglutamic acid residue and to attach a targeting moiety to theaspartic amino acid residue in the second position of ranpirnase,removal of the pyroglutamic acid residue eliminates the bioactivity ofranpirnase.

However, the RNases disclosed in the above-referenced U.S. Pat. No.6,239,257 B1 are not only active against certain human cancer cells, butalso lack “blocked” N-terminals. For this reason, each of these RNasescould be used to make a targeted fusion protein by attaching a targetingmoiety to its N-terminal end.

It would be advantageous to provide methods for manufacturing suchproteins recombinantly.

It would further be advantageous to provide bioactive proteins thatcould be made into targeted fusion proteins.

In accordance with one aspect of the invention, methods are provided forrecombinantly manufacturing the proteins disclosed in U.S. Pat. No.6,239,257 B1.

In accordance with another aspect of the invention, new proteins areprovided that possess activity against human carcinoma cells and thatcan also be manufactured recombinantly. One of the proteins is“cysteinized” to permit easier conjugation to a targeting moiety.

When recombinantly manufactured, one of the proteins disclosed in U.S.Pat. No. 6,239,257 B1 retains its activity against human carcinoma cellseven when a number of different leader sequences are attached to itsN-terminal. The leader sequences form parts of the vector in which theDNA of the protein of interest has been inserted. As will be seen below,there is a compelling body of evidence that such leader sequences donot, when attached to the N-terminal of any one of the family of RNaseproteins disclosed in U.S. Pat. No. 6,239,257 B1, affect the bioactivityof the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the followingexemplary and non-limiting drawings, in which:

FIG. 1 is a flow chart illustrating the process for recombinantlymanufacturing the protein identified as 2325p4 in U.S. Pat. No.6,239,257 B1;

FIG. 2 is a flow chart illustrating the process for recombinantlymanufacturing the protein identified as 2325p6 in U.S. Pat. No.6,239,257 B1;

FIG. 3 is a flow chart illustrating the process for recombinantlymanufacturing the protein identified as 2728 in U.S. Pat. No. 6,239,257B1;

FIG. 4 is a flow chart illustrating the process for manufacturingpET22b-2325p4 DNA;

FIG. 5 is a flow chart illustrating the process for recombinantlymanufacturing the protein identified as 2325p4a in U.S. Pat. No.6,239,257 B1;

FIG. 6 is a flow chart illustrating the process for recombinantlymanufacturing 2325p4-Cys71 protein; and

FIG. 7 is a flow chart illustrating the process for manufacturinghEGF-linker-2325p4-Cys71 fusion protein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

A common procedure is used in the following Examples 1, 2, and 3, whichrelate to recombinant production of proteins identified as 2325p4,2325p6, and 2728 in U.S. Pat. No. 6,239,257 B1. This procedure will bedescribed first at a general level and then in more detail. Thereafter,each Example will be given.

At a general level, fourteen oligonucleotides for each gene (sevenrepresenting the top DNA strand and seven for the bottom DNA strand)were synthesized. The oligonucleotides were cautiously designed so that:

-   -   a) after annealing, complementary oligonucleotides had an        overhang at the 5′ end of each pair, each such overhang being 7        oligonucleotides long; and    -   b) each such overhang had at least three nucleotide mismatches        with the overhang of an unfitting pair of oligonucleotides.

Seven pairs of oligonucleotides, representing both strands of thefull-length gene, were obtained after annealing. The duplexoligonucleotides were ligated in three steps to form full-length DNA ofthe protein of interest. This full-length DNA was then subjected to PCR.The PCR primers were chosen to:

-   -   a) incorporate a XbaI restriction site at the 5′ end of the gene        and a BamHI restriction site at the 3′ end of the gene. These        sites were selected so the DNA could be cloned into a pET-11d        plasmid vector at these sites.    -   b) include a translation initiation codon immediately before the        first nucleotide of the gene.    -   c) incorporate a translation termination codon immediately after        the last nucleotide of the final codon of the gene.        The purified gene thus produced was inserted into a pET11d        plasmid vector between XbaI and BamHI restriction sites. The        insert positive clones were identified and used to express        recombinant protein.

In each instance, the expressed protein had an additional methionineresidue at position −1. This was cleaved in vitro using Aeromonasaminopeptidase to yield the desired protein.

More specifically, in each instance fourteen oligonucleotides weresynthesized and gel purified by Genosys Biotechnologies, Inc. (TheWoodlands, Tex.). Each oligonucleotide was phosphorylated at its 5′ endusing T4 polynucleotide kinase enzyme and its reaction buffer from NewEngland Biolabs, Inc. (Beverly, Mass.). The desired DNA was extractedwith Phenol:Chloroform solution (Eastman Kodak Company, Rochester, N.Y.)and unincorporated rATP was removed by ethanol precipitation.

Each solution of complementary oligonucleotides (20 μg each, for a totalof 40 μg) was mixed and annealed to form duplex oligonucleotides.Annealing was carried out by placing a tube containing the complementaryoligonucleotides in a beaker containing boiling water and thentransferring the beaker to a cold room for approximately 18 hours withgentle stirring.

The annealed duplex oligonucleotides were then agarose gel purifiedusing a Jetsorb DNA extraction kit from Genomed Inc. (Research TrianglePark, N.C.). The duplex oligonucleotides (approximately 10 μg each) weremixed and ligated together in three separate ligation steps at 16° C.for 18 hours using T4 DNA ligase enzyme from New England Biolabs, Inc.(Beverly, Mass.). As above, the DNA in each ligation reaction mixturewas precipitated with ethanol after extracting it with Phenol:Chloroformsolution. This produced full-length double stranded DNA of the proteinof interest.

This product, which was the desired gene, was amplified using PCR andpurified from agarose gel using a Jetsorb DNA extraction kit. Thepurified gene was then digested with XbaI and BamHI restriction enzymesfollowed by its ligation into a pET11d plasmid vector (Novagen) that hadalso been digested with XbaI and BamHI restriction enzyme fromStratagene (La Jolla, Calif.). (It will be understood that the use of apET11d vector, and of XbaI and BamHI restriction sites, is onlypreferred and not necessary. Another vector, and other restrictionsites, could be used instead.)

Then, the ligated reaction mixture was used to transform E. coli strainXL1-Blue (Stratagene) competent cells. The clones were identified forthe insert DNA of the desired protein in the plasmid DNA preparations byrestriction enzyme analysis. The recombinant plasmid DNA was then usedas described below to transform the expression host to express thetarget gene.

E. coli BL21(DE3) competent cells (Novagen, Madison, Wis.) were used asan expression host and transformed with the plasmid DNA. (Anotherexpression host could have been used instead.) The recombinant proteinwas expressed by induction with IPTG. Most of the expressed protein wasfound in the inclusion bodies and some was also present in the solublefraction.

To purify the recombinant protein, the bacterial pellet containing theinclusion bodies was resuspended, sonicated and centrifuged using theprocedure of Schultz and Baldwin (Protein Science 1, 910-916, 1992),modified as discussed below. The inclusion bodies were washed with 50 mMTris-HCl buffer, pH 8.5 containing 300 mM sodium chloride andcentrifuged. The proteins present in the pellet were then denatured with6 M guanidine-HCl in 100 mM Tricine buffer, pH 8.5. Thereafter, theproteins were reduced and fully unfolded by adding 0.1 M reducedglutathione followed by incubation at room temperature under nitrogenfor 3 h. Then, the proteins were refolded by 10 times dilution withnanopure water followed by incubation at 4-5° C. for 18 h. The refoldedprotein was then purified by cation exchange chromatography onSP-Sepharose. The SP-Sepharose column was eluted with a linear sodiumchloride gradient (0-0.3 M) in 0.15 M sodium acetate buffer, pH 5.0.Finally, the homogeneity of the purified proteins was checked by 10-20%SDS-polyacrylamide gel electrophoresis. Although these steps werepreferred to increase the yield of the desired protein, they are notnecessary to the invention and may be omitted.

Finally, as stated above, the initial methionine residue at position-1was cleaved in vitro by Aeromonas aminopeptidase. This produced thedesired protein.

Example 1 Synthesis, Cloning, and Expression of pET1d-2325p4 Plasmid DNA

Example 1 relates to a protein identified as 2325p4 in U.S. Pat. No.6,239,257 B1, which has the amino acid sequence of SEQ ID NO:1 and thenucleotide sequence of SEQ ID NO:2.

In an initial step, oligonucleotides SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,and SEQ ID NO:16 were synthesized and purified as discussed above.

In the next step (shown at the top of FIG. 1 and described in detailabove), pairs of oligonucleotides were mixed and annealed to form duplexoligonucleotides A1, A2, A3, A4, A5, A6, and A7.

These annealed oligonucleotides A1, A2, A3, A4, A5, A6, and A7 were thenagarose gel purified as discussed above. The annealed and purifiedoligonucleotides were then mixed and ligated together in three separateligation steps shown in the center of FIG. 1 using the proceduredescribed above. This produced full-length DNA.

1 μg of the full-length DNA was subjected to PCR with primers SEQ IDNO:3 and SEQ ID NO:16. As discussed above, the primers provide XbaI andBamHI restriction sites permitting the gene to be inserted in a pET11dvector.

The gene of the 2325p4 protein was agarose gel purified as discussedabove. The purified 2325p4 gene was then digested with XbaI and BamHIrestriction enzyme and ligated into a pET11d plasmid vector as discussedabove.

Then, as discussed above, the ligated reaction mixture was used totransform E. coli XL1-Blue competent cells, and the recombinant plasmidpET11d-2325p4 DNA was then used to transform the expression host toexpress the target gene as discussed above.

The expressed protein has the amino acid sequence shown in SEQ ID NO:59,in which an additional N-terminal methionine residue is followed bylysine, the first amino acid of the 2325p4 protein. The N-terminaladditional methionine residue was cleaved as stated above_to yield2325p4 recombinant protein having the amino acid sequence SEQ ID NO:1.

As stated in U.S. Pat. No. 6,239,257 B1, 2325p4 protein inhibited growthof human submaxillary gland carcinoma (A-253) cells and human bladdercarcinoma (T-24) cells.

Example 2 Synthesis, Cloning, and Expression of pET11d-2325p6 PlasmidDNA

Example 2 relates to a protein identified as 2325p6 in U.S. Pat. No.6,239,257 B1, which has the amino acid sequence of SEQ ID NO:17 and thenucleotide sequence of SEQ ID NO:18.

In an initial step, oligonucleotides SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDNO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, and SEQ ID NO:32 were synthesized and purified as discussedabove.

In the next step (shown at the top of FIG. 2 and described in detailabove), pairs of oligonucleotides were mixed and annealed to form duplexoligonucleotides A8, A9, A10, A11, A12, A13, and A14.

These annealed oligonucleotides A8, A9, A10, A11, A12, A13, and A14 wereagarose gel purified as discussed above. The annealed oligonucleotideswere mixed and ligated together in three separate ligation steps shownin the center of FIG. 2 using the procedure described above. Thisproduced full-length DNA.

1 μg of the full-length DNA was subjected to PCR with primers SEQ IDNO:32 and SEQ ID NO:33. As discussed above, the primers provide XbaI andBamHI restriction sites permitting the gene to be inserted into a pET11dplasmid vector.

The double stranded full-length PCR product, namely the gene of the2325p6 protein, was purified from agarose gel and ligated into a pET-11dplasmid vector at XbaI and BamHI restriction site, all using theprocedure discussed above.

Then, using the same procedure described above, E. coli XL1-Bluecompetent cells were transformed and the recombinant plasmidpET11d-2325p6 DNA was used to transform the expression host (E. coliBL21(DE3) competent cells) to express the target gene.

The expressed protein has the amino acid sequence shown in SEQ ID NO:60,in which an additional N-terminal methionine amino acid is followed bylysine, the first amino acid of the 2325p6 protein. The N-terminaladditional methionine residue was cleaved as stated above to yield2325p6 recombinant protein having the amino acid sequence SEQ ID NO: 17.

As stated in U.S. Pat. No. 6,239,257 B1, 2325p6 protein inhibited growthof human submaxillary gland carcinoma (A-253) cells and human bladdercarcinoma (T-24) cells.

Example 3 Synthesis, Cloning, and Expression of pET11d-2728 Plasmid DNA

Example 3 relates to a protein identified as 2728 in U.S. Pat. No.6,239,257 B1, which has the amino acid sequence of SEQ ID NO:34 and thenucleotide sequence of SEQ ID NO:35.

In an initial step, oligonucleotides SEQ ID NO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ IDNO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, and SEQ ID NO:49 were synthesized and purified as discussedabove.

In the next step (shown at the top of FIG. 3 and described in detailabove), pairs of oligonucleotides were mixed and annealed to form duplexoligonucleotides A15, A16, A17, A18, A19, A20, and A21.

These annealed oligonucleotides A15, A16, A17, A18, A19, A20, and A21were agarose gel purified as discussed above. The annealedoligonucleotides were mixed and ligated together in three separateligation steps shown in the center of FIG. 3 using the proceduredescribed above. This produced full-length DNA.

1 μg of the full-length DNA was subjected to PCR with primers SEQ IDNO:33 and SEQ ID NO:49. As discussed above, the primers provide XbaI andBamHI restriction sites permitting the gene to be inserted into a pET11dplasmid vector.

The double stranded full-length PCR product, namely the gene of the 2728protein, was purified from agarose gel and ligated into a pET11d plasmidvector, all using the procedure described above.

Then, using the same procedure described above, E. coli XL1-Bluecompetent cells were transformed and the recombinant plasmid DNApET11d-2728 was used to transform the expression host cell (E. coliBL21(DE3) competent cells) to express the target gene.

The expressed protein has the amino acid sequence shown in SEQ ID NO:61, in which an additional N-terminal methionine amino acid is followedby lysine, the first amino acid of the 2728 protein. The N-terminaladditional methionine residue was cleaved as stated above to yield 2728recombinant protein having the amino acid sequence SEQ ID NO: 34.

As stated in U.S. Pat. No. 6,239,257 B1, 2728 protein inhibited growthof human submaxillary gland carcinoma (A-253) cells and human bladdercarcinoma (T-24) cells.

Example 4 Synthesis and Cloning of pET22b-2325p4 DNA

As stated above, the protein identified as 2325p4 in U.S. Pat. No.6,239,257 B1 has the amino acid sequence of SEQ ID NO:1 and thenucleotide sequence of SEQ ID NO:2. The process for making pET22b-2325p4DNA is illustrated in FIG. 4.

The above-described pET11d-2325p4 plasmid DNA (consisting of 2325p4 DNAcloned in a pET-11d vector) was used as a template for amplificationusing forward and reverse DNA primers in PCR to produce 2325p4 DNA in aform suitable for cloning into a pET22b plasmid between the MscI andBamHI restriction sites.

The forward primer, which is constructed to have SEQ ID NO:50, wasdesigned to incorporate a MscI restriction site at the 5′ end of thegene. The reverse primer, which is constructed to have SEQ ID NO:16, wasdesigned to have a stop codon flanked by a BamHI site at the 3′ end ofthe gene. These primers were used in a single step of PCR amplification.The amplified DNA was then digested with MscI and BamHI restrictionenzyme and cloned into pET22b plasmid digested with MscI and BamHIrestriction enzymes. The newly constructed plasmid was namedpET22b-2325p4 DNA.

Example 5 Synthesis, Cloning, and Expression of pET11d-2325p4a PlasmidDNA

pET11d-2325p4a DNA has been synthesized by replacing the isoleucineresidue at position 44 of pET11d-2325p4 DNA with valine usingsite-directed mutagenesis. 2325p4a protein has the amino acid sequenceof SEQ ID NO:51 and the nucleotide sequence of SEQ ID NO:52.

Primers were designed to generate DNA fragments containing a) an XbaIrestriction site at the 5′ terminus and b) a stop codon flanked by aBamHI site at the 3′ terminus, and mismatched primers were synthesizedto change the isoleucine residue at position 44 to valine. Thefull-length gene of 2325p4a was made in two steps of PCR amplificationsusing a Perkin Elmer DNA thermal cycler, PCR reagents and DNApolymerase.

In the first step of PCR amplification as shown in FIG. 5, two separatePCR reactions were performed using pET11d-2325p4 DNA as a template. Inthe first PCR reaction, amplification was carried out using primers SEQID NO:33 and SEQ ID NO:54 and in the second PCR reaction, amplificationwas carried out using primers SEQ NO ID:16 and SEQ ID NO:53. These twoPCR reactions resulted in two overlapping DNA fragments, both bearingthe same mutation in the overlapping region introduced via primermismatch.

In the second step of PCR amplification, the two overlappinghalf-fragments were mixed together with primers SEQ ID NO:33 and SEQ IDNO:16 to produce full-length 2325p4a DNA containing the desiredmutation. Then, the amplified full-length 2325p4a DNA was gel purifiedand digested with XbaI and BamHI restriction enzymes and subsequentlycloned into pET11d plasmid cut with XbaI and BamHI restriction enzymes.The newly constructed plasmid was named pET11d-2325p4a DNA.

Recombinant 2325p4a protein was expressed and purified using E. coliBL21(DE3) competent cells in the same way as described above in Examples1, 2, and 3. The protein as expressed has the amino acid sequence of SEQID NO: 68, with an initial methionine residue that is cleaved in vitrousing Aeromonas aminopeptidase to yield the protein having the aminoacid sequence SEQ ID NO: 51. This protein is active against A-253 cells.

Example 6 Synthesis, Cloning, and Expression of pET11d-2325p4-Cys71 DNA

Commonly-owned U.S. Pat. No. 6,175,003 B1 discusses the concept of“cysteinizing” therapeutically active RNases. It would be advantageousto “cysteinize” the 2324p4 protein disclosed in the above-referenced'257 patent to facilitate conjugation of a targeting moiety thereto. The2325p4 protein has now been cysteinized by replacing the threonineresidue at position 71 with cysteine using site-directed mutagenesis toform 2325p4-Cys71, which has the amino acid sequence of SEQ ID NO: 55and the nucleotide sequence of SEQ ID NO: 56.

Primers were designed to generate DNA fragments containing a) an XbaIrestriction site at the 5′ terminus and b) a stop codon flanked by aBamHI site at the 3′ terminus, and mismatched primers were synthesizedto change the threonine residue at position 71 to cysteine. Thefull-length gene of 2325p4-Cys71 was made in two steps of PCRamplifications using a Perkin Elmer DNA thermal cycler, PCR reagents andDNA polymerase.

In the first step of PCR amplification as shown in FIG. 6, two separatePCR reactions were performed using pET11d-2325p4 DNA as a template. Inthe first PCR reaction, amplification was carried out using primers SEQID NO:33 and SEQ ID NO:58, and in the second PCR reaction, amplificationwas carried out using primers SEQ NO ID: 16 and SEQ ID NO:57. These twoPCR reactions resulted in two overlapping DNA fragments, both bearingthe same mutation in the overlapping region introduced via primermismatch.

In the second step of PCR amplification, the two overlappinghalf-fragments were mixed together with primers SEQ ID NO:33 and SEQ IDNO:16 to produce full-length 2325p4-Cys71 DNA containing the desiredmutation. Then, the amplified full-length 2325p4-Cys71 DNA was gelpurified and digested with XbaI and BamHI restriction enzymes andsubsequently cloned into pET-11d plasmid cut with XbaI and BamHIrestriction enzymes. The newly constructed plasmid was namedpET11d-2325p4-Cys71 DNA.

Recombinant 2325p4-Cys71 protein was expressed and purified using E.coli BL21(DE3) competent cells in the same way as described above inExamples 1, 2, and 3. The protein as expressed has the amino acidsequence of SEQ ID NO: 69, with an initial methionine residue that iscleaved in vitro using Aeromonas aminopeptidase to yield the proteinhaving the amino acid sequence SEQ ID NO: 55. This protein is activeagainst A-253 cells.

Quite obviously, a targeting moiety can be conjugated to the cysteineresidue at position 71 of the 2325p4-Cys71 protein to direct it to aparticular cell receptor of interest. The selection of an appropriatemoiety is within the skill of a person skilled in the art.

Example 7 Synthesis, Cloning, and Expression ofpET22b-hEGF-linker-2325p4-Cys71 Plasmid DNA

A fusion gene (hEGF-linker-2325p4-Cys71 DNA) cloned in pET22 plasmidvector has been synthesized and expressed. The recombinantly producedhEGF-linker-2325p4-Cys71 fusion protein has the amino acid sequence ofSEQ ID NO:70 and the nucleotide sequence of SEQ ID NO:71.

SEQ ID NO:70 is 176 residues long, and consists of:

-   -   a) the sequence of hEGF protein (residues 1 to 53);    -   b) the sequence of the Linker (residues 54 to 62); and    -   c) the sequence of the 2325p4-Cys71 protein sequence (residues        63 to 176)

The full-length gene of hEGF-linker-2325p4-Cys71 was synthesized asshown in FIG. 7, using three steps of PCR amplification carried outusing a Perkin Elmer DNA thermal cycler, PCR reagents, and DNApolymerase. pET22b-hEGF DNA and pET11d-2325p4-Cys71 DNA were used astemplates for amplification.

In the first step of PCR amplification, the plasmid pET22b-hEGF DNA wasused as a template for amplification using primers SEQ ID NO:72 and SEQID NO:74. The primer of SEQ ID NO:74 has the C-terminal nucleotidesequence of hEGF, followed by the nucleotide sequence of the linker.

In the second step of PCR the plasmid pET11d-2325p4-Cys71 DNA was usedas a template for amplification using primers SEQ ID NO:16 and SEQ IDNO:73. As stated above, the primer of SEQ ID NO:16 was designed togenerate a stop codon flanked by a BamHI site at the 3′ terminus. Theprimer of SEQ ID NO:73 contains the nucleotide sequence of the linker,followed by the N-terminal nucleotide sequence of 2325p4-Cys71 DNA.

These two PCR reactions resulted in two overlapping DNA fragments. Inthe third PCR step, these two overlapping fragments were mixed togetherwith primer SEQ ID NO:72 and SEQ ID NO:16 to produce full-lengthhEGF-linker-2325p4-Cys71 DNA. The amplified full-lengthhEGF-linker-2325p4-Cys71 DNA was agarose gel purified as above, digestedwith BamHI restriction enzyme, and finally ligated into pET22b plasmidcut with MscI and BamHI restriction enzymes.

The newly constructed plasmid was named pET22b-hEGF-linker-2325p4-Cys71DNA.

E. coli BL21(DE3) competent cells were transformed withpET22b-hEGF-linker-2325p4-Cys71 plasmid DNA and the recombinant proteinwas expressed and as in Examples 1, 2, and 3 above. The protein asexpressed has the amino acid sequence of SEQ ID NO: 70. This protein isactive against A-253 cells.

Example 8 Expression of Proteins from pET22b-2325p4 Plasmid

A surprising result occurred when the 2325p4 protein was expressed in E.coli BL21(DE3) competent cells from pET22b-2325p4 plasmid as discussedabove in Example 1. Four separate bioactive proteins were expressed, andall of them were active against A-253 cells. The first of these was the2325p4 protein, which has the amino acid sequence shown in SEQ ID NO:1.

The second protein was the 2325p4 protein preceded by a two residue longleader sequence having the amino acid sequence of SEQ ID NO:62 (thesecond protein therefore has the amino acid sequence of SEQ ID NO:63).The third protein was the 2325p4 protein preceded by a seven residuelong leader sequence having the amino acid sequence of SEQ ID NO:64 (thethird protein therefore has the amino acid sequence of SEQ ID NO:65).The fourth protein was the 2325p4 protein preceded by a twenty-tworesidue long leader sequence having the amino acid sequence of SEQ IDNO:66 (the fourth protein therefore has the amino acid sequence of SEQID NO: 67). Each of these leader sequences is derived from the pelBleader sequence of the pET22b vector.

To a person skilled in the art, the fact that all four of these proteinsremained active is very strong evidence that any protein made up of the2325p4 protein preceded by at least one and at most all of the residuesin the seven residue long leader sequence of SEQ ID NO:64 in order willbe active as well. And, the same is true of any protein made up of the2325p4 protein preceded by at least one and at most all of the residuesin the twenty two residue long leader sequence of SEQ ID NO:66 in order.In other words, since the leader sequences of SEQ ID NO:64 and SEQ IDNO:66 did not affect the activity of the 2325p4 protein, any personordinarily skilled in the art would expect that shortened versions ofthese leader sequences would, when likewise attached at the N-terminalend of the 2325p4 protein, leave the bioactivity of the 2325p4 proteinunaffected.

Furthermore, given that the 2325p6 and 2728 proteins are also activeagainst A-253 and T-24 cells, a person skilled in the art would concludethat adding all or any similarly-shortened shortened part of the SEQ IDNO:64 or the SEQ ID NO:66 leader sequences to the N-terminal end of the2325p4 protein, to the N-terminal end of the 2325p6 protein, or to theN-terminal end of the 2728 protein, would also produce a bioactiveprotein. This is because these proteins are highly homologous and havehighly similar activities against the same cancer cells.

Although one or more preferred embodiments have been described above,the invention is defined only by the following claims:

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